Contamination of maize male-sterile seeds by male-fertile seeds can significantly reduce crop yields. Currently, such contamination can only be detected at the time of pollen maturation. It is then necessary to manually emasculate the maize plants resulting from this contamination. The process must be done within a short and specific time frame, within one week after pollen formation. The task is very expensive and labor-intensive. A typical cost ranges from 3,000 to 4,000 FF (french francs=US$600 to $800) per hectare. In France 60,000 hectares are used to provide sufficient maize seed for planting 3,000.000 hectares of maize production. The cost of production and land area required in the U.S. are proportionate. The price of hybrid seeds is about 20,000 FF (US$4,000) per hectare. This invention""s practical application includes the use of a kit that will allow the PCR amplification of cytotype-specific mtDNA sequences.
One aspect of the invention concerns a polymerase chain reaction (PCR) method that allows one to distinguish and identify without any ambiguity, various male-fertile and male-sterile maize plants. This identification is based on the knowledge of sequences specific to the mitochondrial genome (mtDNA) of the various maize cytotypes. The method can be used to quantify contamination between the various cultivars. The invention also includes the specific sequences of maize mtDNA that have been found to be cytotype-specific.
A specific region in the maize mitochondrial genome has been shown to be rearranged through recombination in four different maize cytotypes: NA and NB (fertiles), S and C (steriles). PCR reactions allow the amplification of DNA fragments of different sizes which are specific to each cytotype. Unidentified cultivars can therefore be identified, and cross-contamination between cultivars quantified.
This invention can resolve two problems:
1) It will identify a maize cultivar cytoplasm whose source is questionable and may be subject to an ownership dispute.
2) It will identify and quantify contamination of fertile seeds within a set of sterile seeds. The quantification of contamination will allow one to estimate the eventual resulting decrease in crop yield so as to better control yields. Knowing how much contamination of fertile seeds is present within a set of male-sterile seeds is very important for seed companies since the beneficial effects of heterosis would be lost resulting in a significant decrease of crop yield. Furthermore certification and commercialization of maize seeds must meet specified quality control standards.
In general, the invention provides novel sequences of mtDNA which are specific for maize cytotypes NA, NB, S and C. Each of the disclosed mtDNA sequences contains one or more subsequences which are unique for the designated cytotype. A method is provided using PCR amplification of the cytotype-specific subsequences to identify the cytotype of a given sample of maize tissue, seed or flour. Further, the method makes it possible to identify the cytotype composition of a sample of mixed cytotypes, for example in seeds or flour. Certain primer sequences for amplifying cytotype specific subsequences are exemplified herein. One skilled in the art will recognize that other primers can be chosen, based upon the disclosed mtDNA sequences, and the detailed conditions for DNA extraction, amplification, and analysis can all be varied within parameters known to those skilled in the art, as may be desired or necessary, depending on the particular needs of those using the method. All such variations will be understood as falling within the scope of the invention.